Methods for making plant protein concentrates

ABSTRACT

Methods of making food-grade plant protein compositions, including yellow pea protein compositions, are provided. The methods use pulses, such as yellow peas, and flours derived therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/871,329 that was filed Jan. 15, 2018, the entire contents ofwhich are hereby incorporated herein by reference.

FIELD OF THE INVENTIONS

The field of this invention is food-grade yellow pea proteinconcentrates prepared from yellow field peas and flours derivedtherefrom, methods of making the concentrates, and food productsprepared using the concentrates.

Some embodiments of the yellow pea protein concentrates arecharacterized by a complete amino acid profile for older children,adolescents, and adults (ages 3 and up), greater than or equal to 23milligrams of sulfur-containing amino acids (cysteine+methionine) pergram of protein, and in vitro digestibility score of 1.00, and a PDCAASvalue of 1.00, including the process to manufacture such pea proteinproducts and food products prepared using such pea protein products.Included in these embodiments are yellow pea protein concentratescontaining at least 27 milligrams of sulfur-containing amino acids(cysteine+methionine) per gram of protein, which provides them with acomplete amino acid profile for children (ages 6 months to 3 years).

BACKGROUND

The market for plant protein concentrates as nutritional supplements islarge and growing. Unlike protein concentrates derived from some otherplants, such as soybeans, protein concentrates derived from yellow fieldpeas have the advantage of being hypoallergenic. Unfortunately, yellowpea protein concentrates having a complete essential amino acid profile,a digestibility score of 1.00, and Protein Digestibility Corrected AminoAcid Score (PDCAAS) of 1.00 are not currently available.

Certain cultivars of yellow peas naturally contain the correct aminoacid profile to meet one of the age categories for a complete amino acidprofile, as published by the Food and Agricultural Organization of theUnited Nations/World Health Organization (FAO/WHO). However, thedigestibility of yellow field peas is relatively low, with typicalDigestibility Scores of 0.61-0.74, primarily due to the presence of lowdigestible proteins (e.g., prolamins and glutelins), fibers, and theinfluence of anti-nutritional factors. (See, Field Peas: ChemicalComposition and Energy and Amino Acid Availabilities for Poultry”,Canadian Journal of Animal Science, 1997 and the Canadian Field PeaIndustry Guide, Third Edition, 2003 and Leterme et al. in the Journal ofthe Science of Food and Agriculture, vol. 53 pp. 107-110 (1990).)Moreover, the processing techniques presently used to extract, isolate,and concentrate pea proteins from yellow peas result in concentratesthat do not have a complete essential amino acid profile, even when theyare derived from yellow pea pulses that may have a complete essentialamino acid profile.

The processes currently being used for the production of proteinconcentrates from yellow field peas and flours produced therefromgenerally include three steps: 1) aqueous protein extraction, 2)separation of water-insoluble materials, and 3) a final separation ofwater-soluble non-protein components from the proteins. The proteinextraction (Step 1) is performed in one of three processes: alkaline pHextraction, wherein the pH of a yellow pea/water slurry is increased byadding sodium, potassium, or calcium hydroxide; fermentation with lacticacid or other bacteria; or salt extraction, wherein a specific ionicstrength of salt is added to take advantage of the salting-in andsalting-out phenomena of proteins. In Step 2, the major portion of thewater-insoluble, non-protein components of starch, crude fiber, sugars,non-starch polysaccharides, oligosaccharides, fat, and ash are removedfrom the protein extracts from Step 1 by separating the insolublematerials from the water phase using physical methods such as settling,decanting, centrifuging, filtrating, or screening to provide aprotein-enriched extract and an insoluble co-product. In Step 3, theprotein-enriched extract obtained in Step 2 is further processed toseparate the water-soluble proteins from other water-soluble non-proteincomponents in the protein-enriched extract. This step is most commonlycarried out using an isoelectric precipitation (also referred to as acidprecipitation).

To address the deficiencies in the essential amino acid profiles of peaprotein concentrates made using conventional processing techniques,producers have resorted to blending together concentrates derived fromdifferent protein sources (with different limiting amino acidcompositions) to create a product which has a blended amino acid profilethat meets the industry standards for a complete amino acid profile. Forexample, typically companies would blend pea and rice proteins together,as rice is high in sulfur-containing amino acids and low in lysine;whereas, pea is high in lysine and low in sulfur-containing amino acids.For example, U.S. patent application publication number 20080206430,“Compositions Consisting of Blended Vegetarian Proteins” identifies aprotein blend of soy, peas, and rice proteins blended in specificproportions to obtain a protein product with a blended amino acid of1.00; even still, this blended formula does not provide a PDCAAS of 1.00because the digestibility of the protein blend was not 1.00.

SUMMARY

Food-grade yellow pea protein concentrates prepared from yellow fieldpeas and flours derived therefrom are provided. Also provided aremethods of making the concentrates and food products that include theconcentrates.

One aspect of the inventions provides yellow pea protein concentratescomprising proteolytically modified yellow pea proteins, wherein theyellow pea protein concentrate is free of proteins other than yellow peaproteins and has a yellow pea protein concentration of at least 70%,based on dry weight, a complete amino acid profile for older children,adolescents, and adults (ages 3 and up), greater than or equal to 23milligrams of sulfur-containing amino acids per gram of protein, an invitro Protein Digestibility of 1.0, and a PDCAAS value of 1.0. Someembodiments of these yellow pea protein concentrates have asulfur-containing amino acid content of 27 milligrams or greater and,therefore, provide a complete amino acid profile for children aged 6months through 3 years. Some embodiments of the yellow pea proteinconcentrates have a yellow pea protein concentration of at least 80%,based on dry weight.

Embodiments of the yellow pea proteins may be hydrolyzed, but aretypically only mildly hydrolyzed, having a Degree of Hydrolysis (DH) ofat least one and/or a DH of less than 15.

Embodiments of the yellow pea protein concentrates include albuminprotein fragments with molecular weights in the range from about 5 kDato about 40 kDa.

Another aspect of the inventions provides food products that includeyellow pea protein concentrates of the type described herein incombination with a beverage and/or a foodstuff. Thus, the food productscomprise a beverage or foodstuff, other than yellow peas; and a yellowpea protein concentrate combined with the beverage or foodstuff.

In various embodiments of the food products, the yellow pea proteinconcentrate has a yellow pea protein concentration of at least 70%,based on dry weight, a complete amino acid profile for older children,adolescents, and adults (ages 3 and up), greater than or equal to 23milligrams of sulfur-containing amino acids per gram of protein, an invitro Protein Digestibility of 1.0, and a PDCAAS value of 1.0. In someembodiments of the food products, the yellow pea protein concentrateshave a sulfur-containing amino acid content of 27 milligrams or greaterand, therefore, provide a complete amino acid profile for children aged6 months through 3 years. Some embodiments of the yellow pea proteinconcentrates contained in the food products have a yellow pea proteinconcentration of at least 80%, based on dry weight. The food product maybe free of plant proteins other than yellow pea proteins and may be freeof any proteins other than yellow pea proteins.

Exemplary food products include non-dairy liquid beverages, non-dairybeverage powders, and non-dairy yogurts.

Another aspect of the inventions provide a method for making a yellowpea protein concentrate from yellow pea particles, the methodcomprising: (a) conducting an alkaline proteolytic extraction on anaqueous slurry of the yellow pea particles; (b) removing water-insolubleproteins and other water-insoluble components from the aqueous slurry toprovide a water-soluble, protein-rich liquid fraction; (c) performing anamylase carbohydrate reaction on the water-soluble, protein-rich liquidfraction to provide a protein-rich, starch converted liquor; (d)separating water-soluble proteins from water-soluble lower molecularweight peptides and water-soluble non-proteins from the protein-rich,starch converted liquor via ultrafiltration using an ultrafiltrationmembrane with a molecular weight cutoff of 5 kDa or smaller to provide aprotein-rich retentate; and (e) removing water from the protein-richretentate to provide a dry yellow pea protein concentrate.

In various embodiments of the methods, the alkaline proteolyticextraction is conducted at a pH in the range from 7.5 to 9.5 for aduration sufficient to provide the yellow pea proteins with a DH ofgreater than zero and less than 15.

In various embodiments of the methods, the aqueous slurry comprises from5 wt. % to 12 wt. % of the yellow pea particles, the yellow peaparticles having a maximum particles size of less than 200 microns; andthe water-soluble, protein-rich liquid has an insoluble solids contentof no greater than 2.0 vol. %.

In various embodiments of the methods, the amylase and glucoamylasereaction (the “amylase/glucoamylase” reaction) is carried out on thewater-soluble, protein-rich liquid at a pH in the range from 5.0 to 6.0to convert starch, dextrins, maltodextrins, and complex sugars intosmaller fragments to allow these to pass through into the permeatefraction of the ultrafiltration process. Previous attempts to utilizelower molecular weight cutoff membranes did not provide a yellow peaprotein concentration of at least 70%, based on dry weight.Implementation of the amylase/glucoamylase reaction was required toreduce the molecular weight of the carbohydrate fraction allowing thepermeation of these carbohydrates through the ultrafiltration membraneduring the ultrafiltration step, allowing for the recovery of a yellowpea protein concentration having a protein concentration of at least80%, based on dry weight. The utilization of an ultrafiltration membranewith a lower molecular weight cutoff improves the recovery of albuminproteins, thereby increasing protein amino acid score.

In various embodiments of the methods, the ultrafiltration is carriedout in a multistage ultrafiltration-diafiltration system that providesthe protein-rich retentate with a yellow pea protein concentration of atleast 70 wt. % of protein, based on a dry weight basis.

Other principal features and advantages of the invention will becomeapparent to those skilled in the art upon review of the followingdrawings, the detailed description, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention will hereafter be describedwith reference to the accompanying drawings, wherein like numeralsdenote like elements.

FIG. 1 is block flow diagram of one embodiment of a process forproducing a protein concentrate from yellow field peas.

FIG. 2 provides the 2011 FAO/WHO recommended amino acid scoring patternsfor Infant (birth to 6 months), Young Children (6 months to 3 years),and Older Children, Adolescents, and Adults.

FIG. 3 shows the essential amino acid analysis and amino acid score of asplit yellow pea raw material, a yellow pea protein concentrate asdescribed in Example 1, and the combined ultrafiltration permeateproduced from these split peas with a comparison to the amino acidreference pattern for children, aged 6 months to 3 years.

FIG. 4 shows a comparison of the pea protein concentrates produced inExample 1, Example 2 (comparative), and five commercially availableyellow pea protein concentrates produced by other pea proteinmanufacturers with the complete essential amino acid requirement forchildren aged 6 months to 3 years, and to the complete essential aminoacid requirement for older children, adolescents, and adults (aged 3years and up).

DETAILED DESCRIPTION

Food-grade yellow pea protein concentrates prepared from yellow fieldpeas and flours derived therefrom are provided. Also provided aremethods of making the concentrates and food products that include theconcentrates. The yellow pea protein concentrates are characterized by ayellow pea protein content of at least 70 wt. % based on dry weightbasis, a high digestibility and a complete essential amino acid profile.

As used herein, the term yellow pea protein concentrate refers to acomposition that is derived from yellow peas and has a yellow peaprotein concentration of at least 70 wt. %, based on a dry weight basis.This definition for yellow pea protein concentrate, as used herein,includes compositions derived from yellow peas having a yellow peaprotein concentration of at least 90 wt. %, which would be generallyrecognized in the field as yellow pea protein isolates. The term yellowpea proteins include both whole proteins and protein fragments thatresult from, for example, cleaving the tertiary structure of wholeproteins.

Various embodiments of the yellow pea protein concentrates arecharacterized by a complete amino acid profile for older children,adolescents, and adults (ages 3 and up), greater than or equal to 23milligrams of sulfur-containing amino acids (cysteine+methionine) pergram of protein, an in vitro Protein Digestibility of at least 0.98, anda PDCAAS value of at least 0.98. This includes embodiments of the yellowpea protein concentrates having an in vitro Protein Digestibility of atleast 0.99 and a PDCAAS value of at least 0.99, and further includesembodiments of the yellow pea protein concentrates having an in vitroProtein Digestibility of 1.0 and a PDCAAS value of 1.0. In some of theseembodiments, the yellow pea protein concentrates are characterized by acomplete amino acid profile for children ages 6 months to 3 years, sincethey contain greater than or equal to 27 milligrams of sulfur-containingamino acids (cysteine+methionine) per gram of protein.

PDCAAS is a method of gauging protein quality. PDCAAS was adopted by theU.S. Food and Drug Administration and the Food and AgriculturalOrganization of the United Nations/World Health Organization in 1993 asthe preferred method to determine protein quality. PDCAAS is calculatedby multiplying the “Protein Digestibility” for a protein composition bythe composition's “Amino Acid Score”. The Federal Register, Jan. 6,1993, Vol. 58, No. 3, P. 2194 provides a standard listing of commonprotein Digestibility Scores, based on animal studies, and pea proteinconcentrate is identified on the Federal Register with a ProteinDigestibility of 0.94.

Protein Digestibility can be analyzed via an in vitro analysistechnique, as described in U.S. Pat. No. 9,738,920. The digestibilityscore derived from this in vitro technique, which involves enzymaticallydigesting a protein containing sample to simulate digestion that wouldoccur inside a mammalian body, is referred to herein as in vitro ProteinDigestibility. The in vitro Protein Digestibility analysis can beperformed commercially by Medallion Labs (General Mills; Minneapolis,Minn.). Medallion Labs provides the in vitro Protein Digestibility as a“ASAP—Quality Score” (Animal-Safe Accurate Protein Quality Score).

The in vitro Protein Digestibility uses enzymatic digestion stepssimilar to stomach and small intestine for protein digestion. For eachcleavage of a protein backbone by an enzyme, an alpha-amino nitrogen(primary amine) is exposed which is reactive to the colorimetricreagent, ninhydrin, and allows for quantification of the digestionreaction. The analysis entails a pepsin digestion at a pH of 2, followedby a trypsin/chymotrypsin digestion at a pH of 7.5, followed by a TCAprecipitation and centrifugation, followed by the reaction of theresulting supernatant with ninhydrin. Quantification is done byabsorbance spectroscopy.

The other component to PDCAAS calculation is Amino Acid Score. The AminoAcid Score compares a protein's eleven Essential Amino Acids: Histidine,Isoleucine, Leucine, Lysine, Methionine+Cysteine (the sulfur-containingamino acids), Phenylalanine+Tyrosine (aromatic amino acids), Threonine,Tryptophan, and Valine, with the recommended essential amino acidprotein profiles of The Food and Agricultural Organization of the UnitedNations/World Health Organization (1985), which vary depending onfactors like age, weight, gender, and other criteria. In 2011, FAOpublished Food and Nutrition Paper #92 “Dietary Protein QualityEvaluation in Human Nutrition” recommended consolidation of the aminoacid pattern to three categories: Infant (birth to 6 months), YoungChildren (6 months to 3 years), and Older Children, Adolescents, andAdults. Table 1 in FIG. 2 provides the 2011 FAO/WHO recommended aminoacid scoring patterns for each of these three classifications. For thepurposes of this disclosure, complete amino acid profiles for a givenage group refer to the complete amino acid profiles published in the FAOpublished Food and Nutrition Paper #92, as published in 2011 and listedin Table 1 of FIG. 2.

The Amino Acid Score compares the above-referenced amino acid patternwhich is appropriate for the age of the individual in order to determineany limiting amino acid(s). The lowest concentration of limiting aminoacid divided by the pattern for that amino acid equals the Amino AcidScore. For example, for most yellow pea protein concentrates that arecurrently available, an upper limit is placed on the Amino Acid Scoredue to insufficient Sulfur-containing Amino Acids (Methionine+Cysteine).If the complete protein requirement for older children through adults isset at 23 mg/g, and a yellow pea protein concentrate only has 16 mg/gsulfur-containing amino acids then the formula to calculate the AminoAcid Score would be 16 mg/23 mg=0.71.

Amino acid contents and Amino Acid Scores for the yellow pea proteinconcentrates are analyzed using Official Method 988.12 (for amino acids,other than tryptophan) or 988.15 (for tryptophan), as published OfficialMethods of Analysis of AOAC International, 20^(th) Edition (2016),Gaithersburg, Md.

The final formula to determine PDCAAS for a protein concentratemultiplies the Amino Acid Score times the Protein Digestibility. Thus,the PDCAAS for a pea protein concentrate having an Amino Acid Score of0.71 and a Protein Digestibility of 0.94 (obtained from the previouslyidentified Federal Register reference) would be calculated as follows:Amino Acid Score 0.71×Protein Digestibility 0.94=0.66 PDCAAS. Commercialpea protein concentrates typically are deficient in sulfur-containingamino acids (Cysteine+Methionine) and/or tryptophan with Amino AcidScores ranging from 0.65 to 0.73 for children aged 6 months to 3 years.

Without intending to be bound to any particular theory of theinventions, the inventors believe that the ability of the presentprocessing methods to provide yellow pea protein concentrates that havean in vitro Protein Digestibility of 1 and a PDCAAS score of 1 can beattributed, at least in part, to the preferential retention of proteins,such as albumins and albumin protein fragments, that have a highsulfur-containing amino acid content and/or a high tryptophan content.This is consistent with the failure of other yellow pea processingmethods to achieve such high Protein Digestibility, sulfur-containingamino acid contents, and PDCAAS scores. For example, the isoelectricprecipitation process that is used in the commercial production of peaproteins captures most of the globulin proteins, but few, if any, of thealbumin proteins in yellow peas. Salt extraction, followed by insolublesseparation and then dialysis or micellization, another widely usedprocessing technology, also captures only a small portion of the albuminproteins from yellow peas. In contrast, the present processes, whichutilize ultrafiltration with select membranes to separate water-solubleproteins from water-soluble non-protein carbohydrates, fats, and ash areable to preferentially capture a major portion of the albumin proteinsin yellow peas by employing a membrane with a molecular weight cutoff ofabout 5 kDa. In addition, the present methods are able to addressproblems that can hinder the use of ultrafiltration for extractingproteins from protein sources, including reduced throughput and foulingof the membranes by residual starch, fiber, and fat in a clarifiedextract (e.g., in an ultrafiltration feed slurry) and a proteinretentate.

Three primary steps in the methods for producing yellow pea proteinconcentrates are an aqueous alkaline protein extraction with proteolyticprotein modification, followed by a separation of water-insolublematerials from the water-soluble materials, including the water-solubleproteolytically modified yellow pea proteins, and the subsequentseparation of water-soluble, non-protein components and lower molecularweight water-soluble proteins from higher molecular weight water-solubleproteins via ultrafiltration.

FIG. 1 shows a block flow diagram of one embodiment of a process forproducing a protein concentrate from yellow field peas. Initially, theyellow peas from the field, which typically have a protein concentrationof about 20 wt. % to about 28 wt. %, are pre-processed 102 by cleaningand dehulling to remove their hulls and foreign material, such as weeds,soil, and seeds, and then breaking them down into halves and smallerpieces. Alternatively, the cleaned dehulled peas may be dry milled intoa pea flour in a pin mill, pulverizer, or hammer mill with particle sizebetween 100 and 200 microns, and such pea flour may be utilized as theraw material for the present processes. Dehulled peas may also beprocessed into an increased protein flour (e.g., ˜50 to 60 wt. % proteinlevel) in a dry milling and air classification process, as analternative to wet milling. In this process, dehulled peas are jetmilled to a fine powder with particle sizes which allow separation withair classification, such that a protein enhanced fraction is separatedfrom a starch fraction. This high protein flour may also be used as araw material for the present process.

The yellow pea particles 103, which may be a flour, are then dispersedin water to form a slurry to which an alkaline protease and a base areadded to carry out an alkaline proteolytic extraction. Optionally, if ayellow pea flour is not used as the raw material, the yellow peaparticle size can be further reduced by wet milling the slurry using,for example, a colloid mill, a disintegrator, a grinder, or other devicecapable of reducing the particle size of the slurry. By way ofillustration, the yellow pea particle size can be reduced, such that theparticles have a mesh size in the range from about 60 to about 150 mesh,and desirably in the range from about 80 to about 150 mesh (about 100 to200 microns). Typical pea particle concentrations in the slurry are inthe range from about 5 wt. % to 15 wt. %, although concentrationsoutside of this range can be used. The slurry is heated at a temperatureand for a period of time to allow the pea particles to undergo analkaline proteolytic protein extraction 104. For example, the slurry canbe heated to a temperature in the range from about 100 to about 150degrees F. for a period of about 10 to about 60 minutes.

Generally, enough base should be added to increase the pH of the slurryto a pH in the range from about 7.5 to about 9.5. Suitable bases includesodium hydroxide, potassium hydroxide, calcium hydroxide, or acombination thereof. The alkaline protease cleaves the open structure ofat least some of the yellow pea proteins and partially hydrolyses theyellow pea proteins and, therefore, the resulting proteins can bereferred to as proteolytically modified yellow pea proteins. Theconcentration of the alkaline protease in the slurry and the duration ofthe hydrolysis are desirably limited, such that a Degree of Hydrolysis(DH) of no greater than 15 is achieved. This includes embodiments inwhich a DH of no greater than 14 is achieved, further includesembodiments in which a DH of no greater than 13 is achieved, stillfurther includes embodiments in which a DH of no greater than 12 isachieved, and yet further includes embodiments in which a DH of nogreater than 10 is achieved. For example, the DH can be in the rangefrom 1 to 14. DH is calculated using the method entitled Degree ofHydrolysis via O-Phthaldialdehyde (the “OPA Method”) with testmethodology published by Genencor International, Inc. (See, Nielsen P.M., Petersen D., and Dambmann C. 2001. Improved Method for DeterminingFood protein Degree of Hydrolysis. Journal of Food Science, Vol. 66, No.5, pp. 642-646.) Generally, a protease concentration in the range fromabout 0.01 wt. % to about 0.1 wt. % and a reaction time in the rangefrom about 1 hour to about 3 hours are sufficient.

A non-pH stat process may be used to allow the pH of the slurry to bereduced naturally as the hydrolysis reaction liberates a hydrogenmolecule with each cut to the protein molecules. As the reactionprogresses, the pH continues to drop and the activity of the enzyme isreduced with this pH drop, and, thus, the hydrolysis is self-limiting toa great extent. The alkaline protease enzymes are desirably of amolecular weight that enables the majority of the enzymes to be removedfrom the final product in the ultrafiltration step that is discussed ingreater detail below.

The main classes of proteins in yellow field peas are albumins (˜18-25%of the total protein), globulins (˜55-80% of the total proteins), andconvicilin (<5% of the total proteins) with minor quantities ofprolamins and glutelins. (See, “Functional attributes of pea proteinisolates prepared using different extraction methods and cultivars”Stone et al. Food Research International 76 (2015) pp. 31-18.) Globulinsare further classified as legumin (11S fraction, 320-400 kDa molecularweight with 6 subunit pairs each containing one acidic 40 kDa and onebasic subunit of 20 kDa), vicilin (7S fraction, 150,000 molecular weightwith three subunits of 50 kDa each containing subunits forpost-translational proteolytic cleavage resulting in fractions of 12-36kDa), and convicilin (70 kDa). “Pea Proteins: A Review of Chemistry,Technology of Production, and Utilization”, Owusu-Ansah et al. FoodReviews International, 7(1), pp. 103-134 (1991) identifies that peaalbumin proteins contain several thousands of specific proteins withmolecular weight profile ranging from 5 to 80 kDa with two predominantalbumins in roughly equal quantities with 24 kDa and 25 kDa molecularweight fractions.

The alkaline protease cleaves the open tertiary structure of the proteinmolecules at specific points, breaking the protein molecules intosmaller protein fragments having lower molecular weights. In particular,this mild proteolytic modification of the proteins reduces the molecularweight of the legumin, vicillin, and convicillin fractions of the yellowpea proteins into more soluble subunit fractions comprising molecularweights in the range of 20 to 70 kDa and reduces the albumins into moresoluble subunits comprising molecular weights of 1 to 40 kDa.

The proteolytic modification of the proteins to smaller molecularweights and subunit size enables the capture all of globulin proteinswithout plugging the downstream ultrafiltration membranes. It alsoallows for the use of a smaller molecular weight cutoff membranes withan acceptable permeation rate that facilitates the capture and retentionof albumin proteins in the final yellow pea protein concentrates. Inaddition, the smaller subunits of protein molecules are more soluble inthe hot alkaline conditions, making them more bioavailable and improvingtheir digestibility, and also have increased permeation rates throughthe ultrafiltration membrane, which is described in greater detailbelow.

Another benefit of the proteolytic modification of the yellow peaproteins is that the hydrolysis of the proteins by the alkaline proteasereduces the emulsification of the native fat globules present at a lowlevel in the raw material. Naturally occurring fat bodies exist(approximately 2.0 wt. % of the raw yellow peas) as reasonably largeglobules surrounded by emulsifying proteins with hydrophobic regions ofthe proteins sticking into the fat globules and the hydrophilic regionsarranging themselves into the water phase. This emulsification of thefats by the yellow pea globulins can prevent, or significantly hinder,the removal of the fats during the separation of the water-insolublecomponents from the water-soluble components and the subsequentseparation of the water-soluble, non-proteins from the water-solubleproteins. As a result, pea protein concentrates produced by conventionalmethods retain the fats that are present in the raw yellow pea material,which typically have fat contents in the range from about 10 wt. % toabout 11 wt. %. In the present methods, the alkaline proteases hydrolyzeserine and/or phenylalanine molecules causing the fats to becomede-emulsified and broken up into smaller globules that are more easilyoxidized to free fatty acids. These degraded and oxidized fats can thenbe removed by the downstream ultrafiltration process. As a result,embodiments of the methods are able to produce yellow pea proteinconcentrates having a fat content, as measured by acid hydrolyzed fatanalyses, of 8 wt. % or lower, including fat contents in the range from6 wt. % to 8 wt. %.

After achieving the aqueous alkaline extraction with proteolytic proteinmodification, water-insoluble materials (a “cake fraction”) areseparated from a protein-rich liquid extract that is enriched insolubilized proteins and other solubilized components, such as sugars.This can be accomplished, for example, using a physical separationtechnique and equipment, such as centrifugation, decanting, filtration,a hydro cyclone, a settling tank, a screening device, or a combinationthereof. The selection of the devices and the number of separation stepsand/or cycles is made such that an efficient separation of theprotein-rich water-soluble fraction and water-insoluble fraction canoccur. Generally, it is desirable to design the separation such that thequantity of water-insoluble material remaining in the water-solublefraction is less than 3% solids by volume. At this low level, a yellowpea extract with acceptable protein concentration can be obtainedwithout significantly plugging the ultrafiltration membranes used in thesubsequent ultrafiltration. The water-insoluble solids thus separatedare a by-product of the process and contain most of the water-insolubleproteins (prolamins and glutelins), fiber, starch, carbohydrates, andother non-protein materials. The removal of these compounds with lowdigestibility are required to achieve a pea protein concentrate withperfect or near-perfect digestibility. Furthermore, any residualinsolubilized proteins, insolubilized carbohydrates, and fat in theprotein-rich extract will tend to plug membranes.

The protein-rich, water-soluble fraction 105 contains a modestcarbohydrate concentration (for example, about 30% carbohydrates)existing as starch, maltodextrins, smaller dextrins, maltose, glucose,sucrose and fibers. The protein-rich, water-soluble fraction 105 isprocessed by adding a bacterial amylase and a glucoamylase 106 at a pH,temperature, and time that is optimal for such enzymes to reduce thesize of the carbohydrate fractions so that these carbohydrates will passthrough into the permeate in the next step of the inventive process.

The resulting protein-rich, starch converted liquor 107 is furtherprocessed via ultrafiltration 108 to separate the water-solublecarbohydrates and other non-proteins and lower molecular weightwater-soluble proteins from a retentate comprising relatively highermolecular weight water-soluble proteins 109. This step can be carriedout in an ultrafiltration/diafiltration (UF/DF) apparatus usingultrafiltration membranes in a multi-stage system at an elevatedtemperature (i.e., at a temperature above ambient, room temperature);typically, a temperature in the range from about 100 degrees F. to about150 degrees F. Using such an apparatus, the protein-rich, starchconverted liquor from the previous step is fed into the first stage ofthe UF/DF system and is separated into a protein-rich retentate streamand a dilute permeate stream containing a small amount of proteins andmost of the water-soluble non-protein components. The ultrafiltrationmembrane passes proteins and other components having molecular weightslower than the molecular weight cutoff of the membrane and retainsproteins having molecular weights higher than the molecular weightcutoff of the membrane. In this way, the ultrafiltration concentratesthe higher molecular weight yellow pea proteins in a retentate. Inparticular, by using an ultrafiltration membrane having a molecularweight cutoff of 5 kDa or lower, in conjunction with the upstreamalkaline proteolytic enzymatic treatment, the present processes have theability to capture all, or substantially all, of the globulins and themajority of the albumin proteins, thereby improving the nutritionalvalue of the yellow pea protein concentrates derived using the presentmethods.

In some embodiments of the methods, ultrafiltration membranes having amolecular weight cutoff of equal to or less than 5 kDa can be used. Suchmembranes include 5 kDa cutoff, 3 kDa cutoff, and 1 kDa cutoffmembranes. For these lower molecular weight cutoff filters, largermolecular weight carbohydrates present in the soluble fraction after theinsoluble separation step are treated with amylase enzymes with theclarified extract to reduce their molecular weights, allowing forgreater protein concentrations with improved permeation rates fromreduced membrane plugging.

Multiple stages of the ultrafiltration can be carried out in order toprovide a retentate with a high protein concentration. By way ofillustration, a first stage of the ultrafiltration can be carried outuntil the UF feed concentration is at least doubled. The first stageretentate then can be diluted with a quantity of water equal to theamount of permeate removed in the first stage. This diluted retentatethen can be fed into a second stage of the UF/DF system and the secondstage can be carried out until the UF feed concentration is at leastdoubled. The second stage retentate then can be diluted with a quantityof water equal to the amount of permeate removed in the second stage.This diluted retentate then can be fed into a third stage of the UF/DFsystem and the third stage can be carried out until the UF feedconcentration is at least doubled. This process can be repeated througha fourth stage, and further stages to achieve a desired yellow peaprotein level of the final retentate. The resulting yellow pea proteinconcentration is desirably at least 70 wt. %, based on the dry weightbasis, but can be higher. For example, some embodiments of the finalultrafiltration retentate have a yellow pea protein concentration of atleast 72 wt. %, at least 75 wt. %, at least 80 wt. %, and at least 85wt. %, based on the dry weight basis.

The number of stages utilized can be selected to optimize the speed ofpermeation with the area of the membranes to provide a desiredthroughput and product protein concentration. A batch or continuousUF/DF system can also provide a high final protein concentration,depending upon the amount of diafiltration water used. The amount ofdiafiltration water used typically will be between about 75 and about200% of the weight of the original UF feed.

The final permeate and the final retentate obtained from the UF/DFsystem with 5 kDa membranes have very different compositions andproperties, including different amino acid profiles. The final retentatehas an amino acid profile in which the concentrations of 6 of the 11essential amino acids are increased when compared to the raw material,and the final retentate also contains 14 wt. % more sulfur-containingamino acids per mg of protein than the raw material. The amino acidprofile of the typical permeate contains a reduction in theconcentrations of 10 of the 11 essential amino acids when compared tothe raw material peas, and the sulfur-containing amino acid levels arereduced by 42 wt. % in the permeate compared to the raw material.Unexpectedly, the process preferentially recovers more of the essentialamino acids present in the raw material, including the sulfur-containingamino acids and permeates fewer of the essential amino acids, includingthe sulfur-containing amino acids, as illustrated in Example 1.

Another benefit of the ultrafiltration process is the removal of lowmolecular weight enzymes, lipoxygenase, phytic acid, saponins, tannins,oligosaccharides, added alkaline protease, free fatty acids, fatdegradation by-products, and/or other soluble compounds which arenaturally present in the raw yellow peas. Some of these compounds areantinutritional factors which can contribute to poor digestibility ofyellow pea proteins. Additionally, several of the aforementionedcompounds contribute to poor organoleptic properties related to yellowpeas and yellow pea proteins, as well as undesirable off-flavors. Invarious embodiments of the UF/DF process greater than 95 wt. % of theoriginal water is removed, containing a similar amount of water-solublenon-protein solids of molecular weight less than 5 kDa.

The present methods are able to produce yellow pea protein concentrateswith greater than 95% solubility. This is due to the removal theinsoluble proteins prior to the ultrafiltration and to the fact that theultrafiltration process maintains the solubility of proteins that areextracted in the alkaline proteolytic extraction in a soluble form. Bycontrast isoelectric precipitation processes create insoluble proteinsas a necessary step to allow for the separation from the non-proteinsoluble materials. The present methods create and maintain thesolubility of the yellow pea proteins and remove many antinutritionalfactors, resulting in the production of yellow pea protein concentrateshaving a digestibility of 1.00.

The final retentate 109 of the multistage UF/DF system can beheat-treated to kill microorganisms and/or pathogens, and the solidslevel of the heat-treated protein-rich final retentate then can beincreased in an evaporator 110 to the desired level to provide yellowpea protein concentrates 111 with different grades. The heat-treated,evaporated final retentate may be further processed by adding differenttypes and quantities of processing aids and ingredients, heating andcooling steps, and other processes to differentiate the final retentateinto dozens of end-use specific products differentiated by productphysical properties, composition, and functionalities. Thesedifferentiated yellow pea protein concentrates can be spray dried intopowders with typical moisture levels of, for example, 4-6 wt. %moisture.

The yellow pea protein concentrates have improved nutritional andfunctional properties which make them excellent ingredients for use inthe protein fortification of foodstuffs and beverages. Foodstuffs andbeverages refers to substances that are suitable for eating or drinkingby humans and/or other animals. The yellow pea protein concentrates canbe added to a wide range of foodstuffs and beverages, including thosethat would not otherwise have a complete amino acid profile, in order toprovide a food product with improved nutritional properties. Forexample, the yellow pea protein concentrates can be included innutritional beverages, bars, and protein powders, replacing both animalproteins (meat, milk, whey, or egg derived) and other allergenisticplant-based proteins such as gluten and soy. The improvement to thenutritional performance is very important because % Daily Value ofprotein claims on the Nutritional Facts panel that is required on everyfood product sold are based on the PDCAAS of the protein source. Riceproteins is another important hypoallergenic plant based protein, butthe PDCAAS of rice protein is 0.45-0.60 with lysine as the deficientamino acid.

Various embodiments of the food products that include the yellow peaprotein concentrates, including those that include the yellow peaprotein concentrates as the sole protein source, provide a non-animal,plant-based, hypoallergenic food product with equal to or greater than23 mg of sulfur-containing amino acids (Cysteine+Methionine) per gram ofprotein, a complete amino acid score for older children (aged 3 yearsand up), adolescents, and adults, an in vitro Protein Digestibility of1.00, and a PDCAAS of 1.00. Included within these embodiments are foodproducts that provide a non-animal, plant-based, hypoallergenic foodproduct with equal to or greater than 27 mg of sulfur-containing aminoacids (Cysteine+Methionine) per gram of protein, a complete amino acidscore for children (aged 6 months to 3 years), an in vitro ProteinDigestibility of 1.00, and a PDCAAS of 1.00.

The yellow pea protein concentrates can be mixed with food-gradeingredients, including but not limited to, those that are GenerallyRecognized as Safe (GRAS) by the U.S. Food and Drug Administration toprovide a solid (e.g., powder) or liquid supplement that can easily bemixed with or used as foodstuffs and beverages. For example, theconcentrates can be mixed with coloring agents (e.g., natural dyes),flavoring agents (e.g., sugar and other natural sweeteners, naturalvanilla flavoring, etc.), plant-based oils (e.g., canola oil, saffloweroil, grapeseed oil, etc.), vitamins, minerals, preservatives,emulsifying agents, thickeners, and the like. The concentrates, alone orin combination with one of more of these additional ingredients, canthen be included in a foodstuff or beverage by methods known in thefield of food and beverage processing, such as simple mixing. The amountof the yellow pea protein concentrate in a food product will depend onthe beverage of foodstuff to which they are added. By way ofillustration only, some food products will include about 1 to about 99wt. % yellow pea protein concentrate. This includes food products thatcontain about 2 to about 50 wt. % yellow pea protein concentrate andfurther includes food products that contain about 5 to about 30 wt. %yellow pea protein.

Certain embodiments of this invention have been utilized to successfullyproduce plant protein concentrates using other pulses such as driedbeans, lentils, peas, and other legumes as raw materials. It isanticipated that the plant-based protein concentrations from chickpeas,lentils, fava (faba) beans, mung beans and others produced utilizing theinvention will have improved compositional and nutritional value whencompared to their raw materials and protein products produced usingconventional methods.

Specific examples of food products can include the yellow peaconcentrates include non-dairy based drinks, non-dairy yogurts, andnon-dairy nutritional beverage powders.

EXAMPLES

Unless otherwise specifically indicated, concentrations recited aspercentages in this disclosure, including these examples, refer toweight percentages.

Example 1

Processing of milled split yellow peas into a pea protein concentratecharacterized by a complete amino acid profile for children (ages 6months to 3 years), a sulfur-containing amino acid content of greaterthan or equal to 27 milligrams per gram of protein, an in vitrodigestibility of 1.00, and PDCAAS of 1.00.

Yellow Field Peas harvested in Saskatchewan were cleaned, dehulled, andsplit in a commercial splitting operation (purchased from Belle Pulses,Donremy, SK, Canada), and then milled to a 120 mesh flour in a nutrimillstone mill with as-is analysis of protein 21.7%, ash 2.5%, andacid-hydrolyzed fat 1.8%. Sixty-five pounds of this flour was slurriedat a solids level of 8.5% into water at 138 degrees F., pH adjusted to9.2 with NaOH (50% concentration), and 29 grams of Alcalase 2.4 Lprotease was added. This protease reaction was maintained at 135 degreeF. for one hour under agitation, and then was pumped to a Sharples P-660horizontal decanter to separate water-soluble from water-insolublematerial present in the protease reacted slurry. The insoluble fractionseparated from the protease slurry had solids analysis of 20.37% andprotein dry basis analysis of 7.6%. The solubles fraction (extract)separated in the decanter was 2.84% solids and dry basis proteinanalysis of 64.7%. The decanter was operated to achieve 0.7%solids-by-volume of the insoluble material remaining in the solublesfraction.

The solubles fraction (extract) from the decanter was further processedusing a Westfalia SB-7 clarifying disk-type centrifuge separating thefinal insoluble material contained in the extract. The extract wasseparated in the clarifier into a sludge fraction containing theresidual insoluble materials and a clarified extract fraction. Theclarified extract fraction contained 3.00% solids and the insoluble spindown analysis was less than 0.1% solids by volume. The protein recoveryin this alkaline proteolytic extraction, followed by a two-stageinsoluble removal, was 77.9%.

The protein-rich, water-soluble clarified extract was heated to 155degrees F. and the pH was adjusted to 6.0 with citric acid (20%concentration). 18 grams of Enzyme Development Corporation alpha amylase2LC and 55 grams of Enzyme Development Corporation glucoamylase L1000were added to the pH adjusted clarified extract and the reaction allowedto continue for 2 hours to convert larger molecular size starch,maltodextrin, and sugars to smaller molecular size to allow them to passthrough the membrane in the ultrafiltration step.

The pH of the protein-rich, starch coverted liquor was adjusted to 7.5with NaOH (50% concentration), and then fed to a four-stageultrafiltration membrane outfitted with a polyethersulfone membrane with5,000 kDa molecular weight cutoff for separation of the larger proteinmolecules from the sugars, ash, and other non-protein components. Themembrane was purchased from Microdyn Nadir and was an 8038 membrane with31 mil spacers and containing 344 square feet of membrane surface. Theamylase reacted clarified extract was processed and recirculated at 150degrees F. until a 2× concentration of the retentate was achieved in thefirst stage of the ultrafiltration separation and then the processingwas stopped (first stage diafiltration is finished). Diafiltration water(fresh, potable R/O water) was added to the first stage retentate in anamount equal to the permeate removed in the membrane from the firststage and the first stage retentate diluted with diafiltration water wasprocessed in a second stage in the same manner as the first stage. Thesecond stage retentate was diluted with diafiltration water in amountequal to the permeate removed in the second stage. This process wasrepeated until four stages have been completed. The retentate from stage4 was not diluted and was further processed as described below. Theamount of diafiltration water consumed was equal to the permeate removedin the four stages. The final protein-rich retentate contained 5.5%solids with a protein concentration of 79.9% dry basis protein. Theaverage permeate solids was 0.26%, and the protein recovery in theultrafiltration/diafiltration step was 83.2%.

The final protein-rich retentate was vat pasteurized at 190 degrees F.for 2 minutes, cooled to 170 degrees F. and fed to and recirculated inan evaporator until a minimum of 16% solids was achieved or two timesthe final ultrafiltration retentate solids level. The product was spraydried to 6.8% moisture in a NIRO atomizing wheel pilot spray drier withinlet temperature of 188 degrees C. and an exhaust temperature of 85degrees C. The final yellow pea protein concentrate was analyzed tocontain 79.9% dry basis protein. 70.0% percent of the protein containedin the raw material was collected in the pea protein concentrateproduced. The flavor of the spray dried pea protein concentrate was verybland and contained none of the traditional ‘pea’ flavor off notes.

Table 2 in FIG. 3 contains the essential amino acid analysis and aminoacid score of the yellow pea protein concentrate produced from the splitpeas in this Example 1 with a comparison to the amino acid referencepattern for a child aged 6 months through 3 years. The amino acidpattern of the yellow pea protein concentrate produced therefrom was notdeficient in any essential amino acid and had an amino acid score of1.00 and is a complete protein source for children aged 6 months through3 years. It is noted that the amino acid score also is 1.00 for olderchildren, adolescents, and adults. The sulfur containing amino acidscontent of the yellow pea protein concentrate was 27.4 mg/g protein.

The amino acid composition of the split pea raw material, the yellow peaprotein concentrate, and a typical permeate is also summarized andcompared in Table 2 in FIG. 3. The retentate is the concentrated liquidpea protein which becomes the final product of this process, and thepermeate contains all the non-protein components and proteins of lessthan 5,000 kDa molecular weight which have been separated from theretentate in the ultrafiltration/diafiltration step. This is the lastseparation step in the inventive process. When the amino acid profile ofthe typical permeate is compared to the split pea raw material aminoacid profile, ten of the eleven essential amino acids in the split pearaw material are reduced by 15% to 45%. At the same time, five of theeleven essential amino acids in the retentate contains an increase of 1%to 21% of the same amino acids compared to the split pea raw material.The ultrafiltration processing step preferentially concentrates ten ofthe eleven essential amino acids present in the raw material includingthe sulfur-containing amino acids. The sulfur-containing amino acids(cysteine+methionine) are increased in this processing step from 24 mgof these amino acids per gram of protein in the raw material up to 27.4mg of these amino acids per gram of protein in the pea proteinconcentrate, a 14% increase.

The pea protein produced in this Example 1 has a sulfur-containing aminoacid analysis of 27.4 milligrams of sulfur-containing amino acids pergram of protein compared to the FAO/WHO reference pattern of 27 mg/g forchildren aged 6 months through 3 years, and the FAO/WHO referencepattern of older children, adolescents, and adults of 23 milligrams pergram.

The digestibility of the pea protein concentrate produced in thisExample 1 was analyzed by General Mills laboratory (Medallion Labs)using the in vitro analysis technique (U.S. Pat. No. 9,738,920) anddetermined to have a digestibility score of 1.00. The protein contains27.4 mg of sulfur-containing amino acids per gram of protein, the aminoacid score of 1.00, and a digestibility of 1.00, and the pea proteinconcentrate produced in Example 1 is a complete protein for olderchildren, adolescents, and adults with a PDCAAS of 1.00.

Comparative Example 2

This example compares the essential amino acid profile, DigestibilityScore, and PDCAAS for a yellow pea protein concentrate made via anisoelectric precipitation, which is similar in concept and results to afermentation process, to the yellow pea protein concentrate produced inExample 1.

Using the same starting raw material as Example 1: Yellow Field Peasharvested in Saskatchewan were cleaned, dehulled, and split in acommercial splitting operation (purchased from Belle Pulses, Donremy,SK, Canada), and then milled to a 120 mesh flour in a nutrimill stonemill with as is analysis of protein 20.7%, ash 2.5%, and acid-hydrolyzedfat 2.5%. Sixty pounds of this flour (which is the same flour used inExample 1) was slurried at a solids level of 10.1% into water at 140degrees F. with pH adjusted to 9.2 with NaOH (50% concentration). Noprotease reaction was performed and the extraction continued for onehour. The extraction slurry was pumped to a Sharples P-660 horizontaldecanter to separate soluble from insoluble material present in theprotease reacted slurry. The insoluble fraction separated from theprotease slurry had solids analysis of 30.27% and protein dry basisanalysis of 4.7%. The solubles fraction (extract) separated in thedecanter was 3.0% solids and dry basis protein analysis of 52.1%. Thedecanter was operated to achieve 8.3% solids-by-volume of the insolublematerial remaining in the solubles fraction.

The solubles fraction (extract) from the decanter was further processedusing a Westfalia SB-7 clarifying disk-type centrifuge separating thefinal insoluble material contained in the extract. The extract wasseparated in the clarifier into a sludge fraction containing theresidual insoluble materials and a clarified extract fraction. Theclarified extract fraction contained 2.94% solids with 52.7% dry basisprotein analysis and the spin down analysis was 2.5% solids by volume.The protein recovery in this alkaline extraction, followed by atwo-stage insoluble removal, was 88.5%.

The pH of the clarified extract was adjusted to the pH of 4.3 withhydrochloric acid (10% concentration) and the precipitated clarifiedextract was fed to a Sharples P-660 decanter to separate precipitatedprotein (curds) from the acid whey. The precipitated clarified curd hada solids level of 6.61% and a protein dry basis of 48.1%, and the wheyhad a solids level of 2.56% and a protein dry basis of 18.4%. Theprotein recovery in this separation was 84.2%, and the overall proteinrecovery was 74.5% of the protein contained in the raw material. Asingle stage separation was performed with a 2 times concentration ofthe precipitate and 65% of the original water was removed resulting in alower final product protein concentration. A second stage washing of theseparated precipitated protein was not performed, and this second stagewould have increased the protein concentration but would increase theamount of proteins (mostly albumin proteins) lost in the whey furtherremoving the sulfur amino acid amino acid concentration in the final peaprotein product.

The final retentate was vat pasteurized at 190 degrees F. for 2 minutes,cooled to 175 degrees F. and fed into and recirculated in an evaporatoruntil 15.6% solids was achieved. The product was spray dried to 4.3%moisture in a NIRO atomizing wheel pilot spray drier with an inlettemperature of 185 degrees C. and an exhaust temperature of 88 degreesC. The final product was analyzed to contain 69.2% dry basis protein.

Table 3 in FIG. 4 contains the essential amino acid analysis and aminoacid score of the pea protein concentrate produced in this Example 2 andcompares it to the amino acid reference pattern for children aged 6months to 3 years, the amino reference pattern for older children,adolescents, and adults, and the essential amino acid profile of the peaprotein produced in Example 1. It is noted that the raw material forExample 1 and Example 2 is the same lot of milled split yellow peas. Theamino acid pattern of the pea protein concentrate produced in Example 2was deficient in the sulfur-containing amino acids with 21.6 mg/gprotein and has an amino score of 0.80. The pea protein product producedin Example 2 had a digestibility of 0.94 and the PDCAAS is 0.75. The peaprotein produced in this Example 2 was deficient in sulfur-containingamino acids and is a not a complete protein source for children aged 6months through 3 years and is not a complete protein source for olderchildren, adolescents, and adults (ages 3 years and up).

Table 3 in FIG. 4 shows a comparison of pea protein concentratesproduced in Examples 1 and 2 and five yellow pea protein concentratesproduced by other pea protein concentrate manufacturers. The yellow peaprotein concentrate produced in Example 1 had an amino acid score of1.00, while the yellow pea protein concentrate of Example 2 had an aminoacid score of only 0.80. It is noted that the PDCAAS of the concentrateof Example 2 is directly comparable to scores calculated for the Pisanneand Puris yellow pea protein concentrates, which are produced using theion exchange or isoelectric precipitation method and are 25-35% belowthe PDCAAS levels of Example 1.

The yellow pea protein concentrate of Example 2, which was produced inthe alkaline extraction, insolubles separation, and isoelectricprecipitation process compares poorly to the alkaline proteolyticextraction, insoluble separation, and ultrafiltration/diafiltrationprocess of this invention, as further illustrated in Example 1. Clearly,the product and process of Example 2 (which is the most widely usedprocess in commercial production) creates a yellow pea proteinconcentration that is deficient in sulfur-containing amino acids withreduced digestibility from the same raw material.

The amino acid score of the pea protein concentrate of Example 2 may beimproved to a complete amino acid profile by the addition of anadditional processing step. The acid whey generated in the separation ofthe precipitated proteins may be further processed using an amylase andglucoamylase carbohydrate reaction followed by a UF/DF system to captureproteins which are present in this acid whey, and these proteinsscrubbed from the acid whey may be added back to the pea proteinproduced in Example 2 before spray drying. The acid whey contains themajority of the albumin proteins present in the raw peas, and combiningthese albumin proteins with the precipitated proteins will result in aproduct with an amino acid score of 1.00. This is an alternateembodiment of the process.

Comparative Example 3

This example compares the essential amino acid profile, DigestibilityScore, and PDCAAS for commercially available yellow pea proteinconcentrates with the pea protein concentrates produced in Examples 1and 2.

Material Specification and Marketing Communication Information Sheetshave been obtained from the five largest commercial yellow pea proteinmanufacturers worldwide. The commercial yellow pea protein manufacturersare listed below, including the location of their respective countriesof manufacture and the type of processing methods utilized in themanufacture of their respective yellow pea protein concentrates:

Company Location Process Pisanne (Cosucre) Netherlands isoelectricprecipitation Puris (World Foods) US isoelectric precipitation RoquetteFrance salt/ion exchange Shungta China fermentation/precipitationJinguan China isoelectric precipitation

Table 3 in FIG. 4 is a summary of the amino acid analysis data takendirectly from their publicly distributed specification sheets of thefive competitive pea protein manufacturing companies listed abovecompared to the essential amino acid analysis of pea proteinconcentrates produced in Examples 1 and 2. Comparing the amino acidanalysis expressed in milligrams per gram of protein, all fivecommercial producers of yellow pea protein concentrates list asulfur-containing amino acid analysis of 16-21 milligrams per gram ofprotein and the two samples prepared in Examples 1 and 2 contain 27.4and 21.6 milligrams per gram of protein. The suggested reference patternfor older child, adolescents, and adults is 23 milligrams per gram ofsulfur-containing amino acids. The suggested reference pattern forchildren aged 6 months through 3 years is 27 mg/g of protein. The peaprotein concentrate produced in Example 1 exceeds the reference pattern,while all of the commercially available pea protein products aredeficient in sulfur-containing amino acids.

The amino acid score is calculated for the yellow pea proteinconcentrates obtained in Example 1, Example 2, and the five largestyellow pea protein concentrate manufacturers and are shown on Table 3 ofFIG. 4. The yellow pea protein concentrate prepared in Example 1 has acalculated amino acid score of 1.00 and the five commercially availableyellow pea protein concentrates have calculated amino acid scores of0.59 to 0.78. The digestibility for the yellow pea protein concentrateof Example 1 is 1.00, while the commercially available yellow peaprotein concentrates cite a digestibility of 0.94, as identified in theFederal Register. The PDCAAS of the yellow pea protein concentrateproduced in Example 1 was 1.00, while the five commercially availableyellow pea protein concentrates have PDCAAS of only 0.65 to 0.73. ThePDCAAS of the yellow pea protein concentrate of Example 1 was a 27% to35% improvement over the five commercially available yellow pea proteinconcentrates, and a 25% improvement when compared to the yellow peaprotein concentrate of Example 2.

Example 4

Preparation of a hypoallergenic, plant based, non-dairy, asepticallyfilled ready-to-drink beverage (RTD). This RTD beverage has a proteinand macro and micro nutrient content level equal to skim milk from cowswith a sulfur-containing amino acid level of 27 mg per gram of protein,amino acid score of 1.00, a Digestibility Score of 1.00, and a PDCAAS of1.00 for children, aged 6 months to 3 years and, as such, also providesa PDCAAS of 1.00 for older children, adolescents, and adults (aged 3years and up).

Formula Ingredient % Pea Protein Concentrate (produced in Example 1)4.1200% Sucrose 2.5500% Canola/Safflower Oil 0.8000% Tricalciumphosphate 0.4770% Locust Bean Gum(TIC Gums) 0.0320% Gellan Gum KelcogelHS-B(CP Kelco) 0.1200% Sea Salt 0.1000% Liquid SunflowerLecithin(Austrade) 0.1400% Vanilla Flavor #113940 (Natural Flavors Inc.)0.0194% Cream Flavor #115666 (Natural Flavors Inc.) 0.0500% VitaminsPackage WE-24994 (Wright Group) 0.0230% Water 91.5686% Total 100.000000%

Batch Mixing Instructions:

Fill high-shear mixer and batch tank with 70% of batch water called informula at less than 60 degrees F. Start recycle pumping loop betweenhigh-shear mixer and batch holding tank. Add yellow pea proteinconcentrate slowly to the water to ensure hydration with low dusting.Add remaining ingredients in the order shown above to high-shear mixer.Premix Gellan Gum and Locust Bean Gum with Tricalcium Phosphate beforeadding. Continue to mix for 15 minutes and recycle product from batchtank through high-shear mixer. Adjust solids as needed with waterwithheld from batch (approximately 21.56% of formula). Product mixtureshould be continually agitated to prevent settling of TricalciumPhosphate.

Processing Instructions:

Process Product Aseptically using steam injection according to FDAaseptic process filing (293° F. for 9 seconds). Cool to less than 180°F. and homogenize at 3500 psi combined 1^(st)/2^(nd) stage beforethermal processing/sterilization. Continuously agitate product insterile holding tank to prevent settling. Package aseptically insterilized cartons with Nitrogen headspace gas.

Finished Product Solids Target 8.13%±0.10%

100% of the ingredients in the RTD formula in Example 4 are plant-basedand no animal based ingredients are used in the preparation. 100% of theRTD protein source is a pea protein concentrate produced according toExample 1, which has a calculated PDCAAS of 1.00. Accordingly, the RTDproduced in Example 4 is a hypoallergenic, cholesterol-free beveragewith 27 mg of sulfur-containing amino acids per gram of protein, anamino acid score of 1.00, a Digestibility Score of 1.00, and a PDCAAS of1.00 for children, aged 6 months to 3 years and for older children,adolescents, and adults (aged 3 years and up).

Example 5

Preparation of a hypoallergenic, plant based, non-dairy, asepticallyfilled, Greek-style yogurt. This cholesterol-free yogurt has a proteinlevel similar to Greek-style low fat yogurt produced from cow's milk inmacro and micro nutrient content. The yogurt has a sulfur-containingamino acid content of 27 mg per gram of protein, an amino acid score of1.00, a Digestibility Score of 1.00, and a PDCAAS of 1.00 for children,aged 6 months to 3 years and for older children, adolescents, and adults(aged 3 years and up).

Formula Ingredients % Pea Protein Concentrate (produced in Example 1)10.00% Sugar 6.50% Calcium Chloride 0.25% Wright vitamin mineral blend0.4 Active yogurt cultures 0.05 Water 82.80 Total 100.00% FruitPreparation, 20 brix 12.50%

Batch Mixing and Packaging Instructions

Dry blend pea protein, sugar, and vitamin mineral blend. Heat water to100 degrees F. and mix dry blended powders into water in a well agitatedliquid blender. When fully dispersed, heat to 140 degrees and homogenizein a two-stage homogenizer with 2000 psi first stage and 500 psi secondstage. Heat the homogenized mixture to 165 degrees and hold for oneminute, then cool to 140 degree F. Under good agitation, slowly addcultures and dilute calcium chloride. Just prior to packaging, combinefruit preparation with homogenized yogurt by mixing 87.5% of the yogurtmix with 12.50% of the fruit preparation and package aseptically inplastic cups.

100% of the ingredients in the yogurt produced in Example 5 areplant-based and no animal based ingredients are used in the preparation.100% of the protein source in the Greek-style yogurt is a yellow peaprotein product concentrate according to Example 1. Accordingly, theGreek-style yogurt produced in Example 4 is a cholesterol free,hypoallergenic, Greek-style yogurt with a sulfur-containing amino acidlevel of 27 mg per gram of protein, an amino acid score of 1.00, aDigestibility Score of 1.00, and a PDCAAS of 1.00 for children, aged 6months to 3 years, and for older children, adolescents, and adults (aged3 years and up).

Example 6

Preparation of a hypoallergenic, plant based, non-dairy, lactose-freenutritional beverage powder for toddlers aged 3-5 years. Thischolesterol-free toddler beverage has a protein level similar to toddlerformula produced from cow's milk in macro and micro nutrient content.The beverage powder has a sulfur-containing amino acid level of 27 mgper gram of protein, an amino acid score of 1.00, a Digestibility Scoreof 1.00, and a PDCAAS of 1.00 for children, aged 6 months to 3 years andfor older children, adolescents, and adults (aged 3 years and up).

Formula Dry Solids Ingredients % Dry % of liquid mix Tapioca SyrupSolids 39.3% 15.72% High Oleic Sunflower Oil 28.5% 11.40% Pea ProteinConcentrate (produced in Example 1) 20.4% 8.16% Sugar 8.5% 3.40% WrightVitamin Mineral & Oil Blend 3.8% 1.52% Sunflower Lecithin 0.7% 0.28%Salt 0.2% 0.08% Natural Vanilla Flavor 0.075% 0.003% Water — 60.0%Totals 100.00% 100.00%

Mixing and Processing Instructions

Water at 125 degrees F. is put into a liquid blender and the yellow peaprotein concentrate is added under agitation and mixed for 20 minutes toallow the proteins to solubilize. After protein solubilization, thecarbohydrates (tapioca solids and sugar) are added to the liquid proteinmix and held under agitation for 10 minutes after the last addition toallow mixing. The Sunflower Oil and Lecithin are added and the mixtureis heated to 145 degrees F. The Vitamin Mineral Mix, salt, and flavorsare added and the product is immediately homogenized in a two-stagehomogenizer at 2000 psi first stage and 500 psi second stage. Thehomogenized mixture is heated to 200 degrees F., held for 30 seconds,cooled to less than 150 degrees F., and is spray dried to a fine powderwith less than 4.0% moisture in a tall form spray drier. The toddlerpowder is packaged in 1-2-pound cans or plastic bottles. 40 grams oftoddler beverage powder is stirred into 250 ml of room temperature waterbefore consumption by an older child 3 years old and up.

Preparation of a hypoallergenic, plant based, non-dairy, lactose-freenutritional beverage for toddlers aged 3-5 years is accomplished. Thischolesterol-free toddler beverage has a protein level similar to toddlerformula produced from cow's milk in macro and micro nutrient content.The beverage has an amino acid score of 1.00, a Digestibility Score of1.00, and a PDCAAS of 1.00.

100% of the ingredients in the toddler beverage powder produced inExample 6 are plant-based and no animal based ingredients are used inthe preparation. 100% of the protein source in the toddler beveragepowder is a yellow pea protein concentrate produced according toExample 1. Accordingly, the toddler beverage powder produced in Example6 is a cholesterol free, lactose-free, hypoallergenic, toddler beveragepowder with a sulfur-containing amino acid score of 27 mg per gram ofprotein, an amino acid score of 1.00, a digestibility of 1.00, and aPDCAAS of 1.00 for children, aged 6 months to 3 years, and for olderchildren, adolescents, and adults (aged 3 years and up). 250 ml of thereconstituted toddler beverage powder contains 25% of the RDA ofprotein, vitamins, and minerals for an older child of 3-5 years old.

The word “illustrative” is used herein to mean serving as an example,instance, or illustration. Any aspect or design described herein as“illustrative” is not necessarily to be construed as preferred oradvantageous over other aspects or designs. Further, for the purposes ofthis disclosure and unless otherwise specified, “a” or “an” means “oneor more.”

The foregoing description of illustrative embodiments of the inventionhas been presented for purposes of illustration and of description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed, and modifications and variations are possible inlight of the above teachings or may be acquired from practice of theinvention. The embodiments were chosen and described in order to explainthe principles of the invention and as practical applications of theinvention to enable one skilled in the art to utilize the invention invarious embodiments and with various modifications as suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto and theirequivalents.

What is claimed is:
 1. A method for making a plant protein composition,the method comprising: (a) providing an aqueous slurry comprisinghydrolyzed water-soluble and water-insoluble pulse proteins; (b)removing the water-insoluble pulse proteins and other water-insolublecomponents from the aqueous slurry to provide a water-soluble,protein-rich liquid fraction comprising both hydrolyzed globulinproteins and hydrolyzed albumin proteins; (c) separating water-solublepulse proteins in the protein-rich liquid fraction from water-solublelower molecular weight peptides and water-soluble non-proteins in theprotein-rich liquid fraction via ultrafiltration using anultrafiltration membrane that concentrates hydrolyzed globulin proteinsand hydrolyzed albumin proteins in a protein-rich retentate; and (d)removing water from the protein-rich retentate to provide a dry plantprotein composition having a pulse protein concentration of at least70%, based on a dry weight basis.
 2. The method of claim 1, wherein thewater-soluble pulse proteins in the aqueous slurry and in the dry plantprotein composition have a degree of hydrolysis of greater than zeropercent and less than 15 percent.
 3. The method of claim 2, wherein thewater-soluble pulse proteins in the aqueous slurry have a degree ofhydrolysis of greater than 1 percent, but no greater than 12 percent. 4.The method of claim 1, wherein the aqueous slurry comprises from 5 wt. %to 12 wt. % pulse particles, the pulse particles having a maximumparticle size of less than 200 microns; and the water-soluble,protein-rich liquid fraction has an insoluble solids content of nogreater than 2.0 vol. %.
 5. The method of claim 1, wherein the step ofproviding the aqueous slurry comprising the hydrolyzed water-soluble andwater-insoluble pulse proteins comprises conducting an alkalineproteolytic extraction and hydrolysis on an aqueous slurry of pulseparticles.
 6. The method of claim 1, wherein the ultrafiltrationmembrane has a molecular weight cutoff of 5 kDa or smaller.
 7. Themethod of claim 1, wherein the pulse proteins comprise yellow peaproteins and the plant protein comprises yellow pea protein.
 8. Themethod of claim 1, wherein the pulse proteins are selected from chickpeaproteins, lentil proteins, fava bean proteins, and mung bean proteinsand the plant protein is selected from chickpea protein, lentil protein,fava bean protein, and mung bean protein.
 9. The method of claim 1,wherein the dry plant protein has a solubility of at least 95%.
 10. Themethod of claim 1, further comprising performing an amylase carbohydratereaction on the water-soluble, protein-rich liquid fraction afterremoving the water insoluble proteins and other water insolublecomponents from the aqueous slurry, but before separating thewater-soluble proteins in the protein-rich liquid fraction fromwater-soluble lower molecular weight peptides and water-solublenon-proteins in the protein-rich liquid fraction.
 11. The method ofclaim 10, wherein the water-soluble pulse proteins in the aqueous slurryhave a degree of hydrolysis of greater than zero percent and less than15 percent.
 12. The method of claim 11, wherein the water-soluble pulseproteins in the aqueous slurry have a degree of hydrolysis of greaterthan 1 percent, but no greater than 12 percent.
 13. The method of claim10, wherein the aqueous slurry comprises from 5 wt. % to 12 wt. % pulseparticles, the pulse particles having a maximum particle size of lessthan 200 microns; and the water-soluble, protein-rich liquid fractionhas an insoluble solids content of no greater than 2.0 vol. %.
 14. Themethod of claim 10, wherein the amylase carbohydrate reaction is carriedout on the water-soluble, protein-rich liquid fraction at a pH in therange from 5.0 to 6.0 and results in the conversion of starch, dextrins,maltodextrins, and complex sugars into smaller fragments that passthrough into a permeate fraction of the ultrafiltration process.
 15. Themethod of claim 1, wherein the plant protein composition compriseslegumin, vicilin, and convicilin proteins having molecular weights inthe range from 20 kDa to 70 kDa and albumin proteins having molecularweights in the range from 1 kDa to 40 kDa.
 16. The method of claim 1,wherein the dry plant protein composition has a pulse proteinconcentration of at least 80%, based on a dry weight basis.
 17. Themethod of claim 1, further comprising heat-treating the protein-richretentate to kill microorganisms, pathogens, or both.
 18. The method ofclaim 1, further comprising adding the dry plant protein composition toa foodstuff.
 19. The method of claim 1, further comprising adding thedry plant protein composition to a beverage.
 20. The method of claim 1,further comprising mixing the dry plant protein composition with acoloring agent, a flavoring agent, a plant-based oil, vitamins,minerals, preservatives, or emulsifying agents.